UNIVERSITI PUTRA MALAYSIA EXTRACTION OF NATURAL PIGMENTS FROM HYLOCEREUS POLYRHIZUS GROWN IN MALAYSIA NASSIM NADERI FSTM

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1 UNIVERSITI PUTRA MALAYSIA EXTRACTION OF NATURAL PIGMENTS FROM HYLOCEREUS POLYRHIZUS GROWN IN MALAYSIA NASSIM NADERI FSTM

2 EXTRACTION OF NATURAL PIGMENTS FROM HYLOCEREUS POLYRHIZUS GROWN IN MALAYSIA NASSIM NADERI MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA 2008

3 EXTRACTION OF NATURAL PIGMENTS FROM HYLOCEREUS POLYRHIZUS GROWN IN MALAYSIA By NASSIM NADERI Thesis submitted to the school of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of Master of Science May 2009 ii

4 DEDICATION This thesis is dedicated to My parents Who taught me how to work hard and never give up my dreams & Who's loving ways have no bound. iii

5 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science EXTRACTION OF NATURAL PIGMENTS FROM HYLOCEREUS POLYRHIZUS GROWN IN MALAYSIA By NASSIM NADERI May 2009 Chairman: Faculty: Professor Mohd Yazid Abd. Manap, PhD Food Science and Technology The use of natural colors in food is gaining importance because the number of synthetic colorants approved for use in food is legislatively restricted. There is only one type of betacyanin i.e. betanin has been approved as natural food colorant (E-162) and almost entirely obtained from the red beet. Thus, in this study Hylocereus polyrhizus fruit, as an alternative source of betacyanin pigments was investigated to meet the ever-increasing commercial demand. Two solvent extraction systems were investigated to determine the efficiency of solvents in recovering color pigments from pulp of Hylocereus polyrhizus fruit. Individual betacyanin pigments were identified according to High-performance liquid chromatographic analysis. Quantitative and qualitative differences were determined in betacyanin composition of each concentrated extract. Betanin, phyllocactin, hylocerenin and their respective isomers with different ratios were the major betacyanin components of Hylocereus polyrhizus fruits. From solvent system selection, the aqueous ethanolic system was found more effective than ethanol in recovering betacyanins. The alteration of betanin:phyllocactin peak area ratio in iv

6 ethanolic assay revealed the lower stability of phyllocactin that resulted deacylation and betanin formation. Enzymatic treatment method was optimized by means of highest yield of pigment in concentrated extract. Betacyanin retention was monitored in a range of different enzyme dosage (0.1%-2% w/v). The color characteristic of concentrated extracts from solvent extraction assays and enzymatic treatment were compared and monitored along with red beet colorant by using CIELAB system. Chroma (C * ) value was strongly influenced by applied extraction methods. The two extracts obtained from the pulp of fruits by solvent extraction assay provided color tint (h o ) close to the red beet color. Tonality of color extracts from the skin and enzyme-treated shifted into an intense purple color and due to their chroma value displayed darker color. These findings revealed that composition of betacyanin pigments in Hylocereus polyrhizus fruit displayed wider color spectrum compare to red beet. Application of betacyanins in Hylocereus polyrhizus fruit could have considerable potential for development to be employed in the food industry. v

7 Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains PENGEKSTRAKAN PIGMEN- PIGMEN SEMULAJADI DARIPADA HYLOCEREUS POLYRHIZUS YANG DITANAM DI MALAYSIA Oleh NASSIM NADERI May 2009 Pengerusi : Fakulti : Professor Mohd Yazid Abd. Manap, PHD Sains Makanan dan Teknologi Pengunaan pewarna semulajadi semakin mendapat perhatian kerana bilangan pewarna sintetik yang diluluskan oleh pihak berkuasa adalah terhad. Hanya sejenis betacianin iaitu betanin telah dilusluskan sebagai pewarna makanan semulajadi (E-162) dan ianya didapati daripada ubi merah. Maka, kajian dengan menggunakan buah-buahan Hylocereus polyrhizus sebagai sumber gantian bagi pigmen betacyanin telah dijalankan untuk menemui keperluan komersial yang semakin meningkat. Dua ujian pengekstrakan pelarut terpilih telah dijalankan bagi menentukan kecekapan pelarut dalam pemulihan pigmen warna daripada pulpa Hylocereus polyrhizus. Individu pigmen betacianin dicam berasaskan kepada Analisis kromatografi cecair berprestasi tinggi. Perbezaan di antara kuantitatif dan kualitatif telah dipertahatikan bagi komposisi betacianin dalam setiap kepekatan ekstrak. Betanin, phyllocactin, hilocerenin dan isomernya dengan nisbah yang berbeza merupakan komponen betacianin yang utama bagi buah-buahan Hylocereus polyrhizus. Daripada pilihan sistem pelarut, larutan etanol yang dikaji didapati lebih vi

8 berkesan dalam memulihkan betacianin berbanding dengan etanol. Pengubahsuaian kawasan nisbah tertinggi betanin:pilocactin dalam etanol yang dikaji menunjukkan kestabilan pilocactin yang rendah telah menyebabkan pembentukkan deacylation and betanin. Kaedah rawatan dengan enzim adalah dioptimumkan dengan cara hasil tertinggi pigmen dalam ekstrak pekat. Pengekalan Betasianin diawasi dalam satu lingkungan dos enzim yang berbeza (0.1%-2% w/v). Ciri warna kepekatan yang diekstrakkan dari ujian pengekstrakan pelarut dan rawatan enzim telah dibandingkan dan diperhatikan bersama dengan pewarna merah ubi dengan menggunakan Sistem CIELAB. Nilai Kroma (C * ) amat dipengaruhi oleh kaedah-kaedah pengekstrakan yangdigunakan. Dua hasil ekstrak yang didapati daripada pulpa buah-buahan oleh ujian pengekstrakan pelarut membekalkan warna (h o ) yang hampir kepada warna ubi merah. Kualiti nada warna yang diekstrakkan dari kulit dan enzim yang dikaji menunjukkan satu warna ungu yang tumpu dan disebabkan oleh nilai kromanya menunjukkan warna yang lebih gelap. Penemuan ini menunjukkan komposisi pigmen betasianin dalam Hylocereus polyrhizus mempamerkan spektrum warna lebih luas berbanding dengan ubi merah. Aplikasi betacianin dalam Hylocereus polyrhizus akan mempunyai potensi yang tinggi untuk digunakan dalam industri makanan. vii

9 ACKNOWLEDGEMENTS A word of thanks is given to GOD, the source of all knowledge, by whose abundant grace this work came to fruition. I would like to begin by thanking Professor Dr. Mohd Yazid Abd Manap, my thesis supervisor. Your guidance encouragement and faith in my success meant more to me than you can imagine. It will always be appreciated. My sincere thanks go to Professor Dr. Hasanah Mohd Ghazali for accepting to be expert on the thesis committee and her important suggestions and her fruitful discussions. I strongly appreciated the support, advice, freedom and the confidence they granted me during the last two years. I would like to thank all members of the Biochemical lab for their technical help, suggestions and the pleasant atmosphere. Finally, my thoughts go to my parents for their essential logistical help, supports, encouragement and their love. viii

10 I certify that an Examination Committee has met on to conduct the final examination of Nassim Naderi on her Master of Science thesis entitled Extraction of Natural Pigments from Hylocereus polyrhizus grown in Malaysia in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations The Committee recommends that the student be awarded the relevant degree. Members of the Examination Committee were as follows: Name of Chairperson, PhD Title: Assoc. Prof. Badlishah Sham Baharin Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Name of Examiner 1, PhD Title: Assoc. Prof. Azizah Abdul Hamid Faculty of Food Science and Technology Universiti Putra Malaysia (Internal Examiner) Name of Examiner 2, PhD Title: Prof. Dr. Nazamid Saari Faculty of Food Science and Technology Universiti Putra Malaysia (Internal Examiner) Name of External Examiner, PhD Title: Name of Faculty: Name of University: (External Examiner) BUJANG KIM HUAT, PHD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: ix

11 This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of Supervisory Committee were as follows: Mohd Yazid Abd. Manap, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Hasanah Mohd. Ghazali, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Member) HASANAH MOHD. GHAZALI, PHD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: x

12 DECLARATION I hereby declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution. NASSIM NADERI Date: xi

13 TABLE OF CONTENTS DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL SHEETS DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATION Page iii iv vi viii ix xi xvi xviii xx CHAPTER I INTRODUCTION 1 II LITERATURE REVIEW 2.1 Physical basis of color Physiology and attributes of color Measurement of color CIE system of colorimetry Uniform color space and color differences formula Natural food color market Color additives Synthetic food colorants Natural food colors Natural identical food colors Coloring food stuffs Betalain pigments Red beet (Beta vulgaris L. ssp. vulgaris Garden Beet Group ) Amaranthaceae plants (Amaranthus & Celosia) 30 xii

14 2.7.3 Cactus fruits Hylocereus polyrhizus fruits Habitant Potential uses Extraction of betacyanin pigments from plant tissues Detection of pigments Tracking color and pigment changes 40 III PHYSICAL ATTRIBUTES AND CHEMICAL COMPOSITION OF SKIN AND PULP OF HYLOCEREUS POLYRHIZUS 3.1 Introduction General Materials Plant material Chemicals Methods Sample preparation Chemical and physical analysis Pigment extraction Betacyanin content determination HPLC characterization of betacyanins Color analysis Statistical analysis Results and discussions Characterization and chemical composition HPLC analysis to determine the betacyanin pattern of 56 Skin and pulp, total pigment content and color characteristic 3.5 Summary 64 xiii

15 IV EFFECT OF DIFFERENT SOLVENTS IN EXTRACTION AND BETACYANIN CONSTITUENTS OF HYLOCEREUS POLYRHIZUS 4.1 Introduction Material and methods Plant material and chemicals Solvent and reference substances Preparation of the concentrated betacyanin extract Photometric quantification of total betacyanins High-Performance Liquid Chromatography (HPLC)- 70 UV Visible Detector Analysis Other analytical measurements Statistical analysis Results and discussions Isolation of the concentrated betacyanin extracts by 72 solvent system selection Pigment characterization and betacyanin retention in concentrated extracts Summary 81 V EFFECT OF ENZYME TREATMENT ON THE YIELD OF BETACYANIN PIGMENTS AND CONSTITUENTS 5.1 Introduction Materials and methods Plant material Chemical and standard Enzymatic treatment Preparation of the concentrated betacyanin:solvent 86 system Determination of sugars by HPLC analysis HPLC conditions for organic acids Betacyanin quantification Determination of betacyanin retention by HPLC 89 analysis xiv

16 5.2.9 Statistical analysis Results and discussion Enzymatic treatment Monitoring matrix constituent in concentrated 92 betacyanin extracts by applying different dosage of enzyme & solvent extraction Individual betacyanin patterns in concentrated 95 betacyanin extract for enzyme screening 5.4 Summary 104 VI VII COLOR EVALUATION OF CONCENTRATED BETACYANIN EXTRACTS FROM HYLOCEREUS POLYRHIZUS IN COMPARISON WITH RED BEET COLOR 6.1 Introduction Material and methods Plant material and chemicals Isolation of concentrated extracts Spectral measurement Color measurement by CIEL * a * b * system Statistical analysis Results and discussions Spectra characteristic of each colorant Color characteristic of concentrated natural extracts Visual impact of hue and chroma in color differences 118 of concentrated extracts 6.4 Summary 124 SUMMARY, CONCLUSION AND RECOMMENDATION 7.1 Summary Conclusion and recommendation 132 REFERENCES 135 APPENDICES 144 PUBLICATION AND CONFERENCES FROM THE THESIS 152 BIODATA OF STUDENT 153 xv

17 Table LIST OF TABLES Page 2.1 Natural red food color market share by product Natural food color market Caryophyllales and distribution of betalains Main betacyanins and betaxanthins in edible plant source of betalains Summary of external factors and pigment-specific that affect betacyanin stability Grading system of red-purple Hylocereus polyrhizus in Malaysia Chemical composition of skin and pulp of Hylocereus polyrhizus fruits in Malaysia Total contents and relative concentrations (expressed as percentage of the total HPLC peak area) of betacyanins found in analyzed skin and pulp of Hylocereus polyrhizus Relative concentration (Expressed as the percentage of the total HPLC peak area) of the six betacyanins found in analyzed H. polyrhizus from Malaysia and Israel 60 xvi

18 3.5 Chromatic parameters of the extracted fruit pulp and skin obtained from Hylocereus polyrhizus fruits Characteristic of liquid concentrated extracts using solvent assays Pigment characteristics of concentrated extract using organic and aqueous organic extraction assay and betanin/isobetanin, betanin/phyllocactin peak area ratios at 540 nm in H. polyrhizus pigment preparation Concentration ranges and calibration equations of reference compounds used for calibration of the HPLC analysis HPLC qualitative and quantitative data of sugars & organic acids in concentrated betacyanin extracts from H. polyrhizus fruit pulp, applying different extraction methods Relative chromatogram areas and betacyanin ratios of major betacyanins in concentrated color extracts from H. polyrhizus fruits Betacyanin retention in extracted colors obtained through enzymatic treatments monitored at λ max by spectrophotometric & by HPLC analysis Chromatic parameters in concentrated betacyanin from H. polyrhizus using three different solvent systems 116 xvii

19 Figure LIST OF FIGURES Page 2.1 The electromagnetic spectrum. (A)Spectral power distribution of light (B)Visible spectra Three-dimensional color system (A) How hue, saturation (chroma), and lightness are related to each other in a three dimensional color system. (B) A color space based on hue, chroma and lightness uses cylindrical coordinates CIE L*a*b* color space- When a color is expressed in CIE L*a*b*, L* defines lightness; a* denotes the red/green value; and b* the yellow/blue value. This three-dimensional space provided a logical framework within which the relationship between two or more colors can be calculated Chemical structure of betalamic acid (A) the main chromophore of betalains. Predominant betaxanthins (e.g. indicaxanthin from Opuntia ficus-indica)(b), betacyanin (e.g. betanin from Beta vulgaris)(c) Structures of betanin and isobetanin Two year old Hylocereus polyrhizus plant growing on trellis, modern agricultural farm, Kluang, Johor, Malaysia (Oct, 2007) Hylocereus polyrhizus fruits (Grade B: gr)-variety grown in Kluang, Johor, Malaysia Chemical structures of nonacylated betacyanins in fruits from Cactecea: (A) Betanin, Isobetanin and acylated betacyanins: (B) Phyllocactin, Isophyllocactin (C) Hylocerenin, Isohylocerenin 35 xviii

20 3.1 Chromatogram registered at 540 nm corresponding to an extract obtained from skin and pulp of fresh Hylocereus polyrhizus fruits HPLC pattern of extracted betacyanins from fruit pulp of Hylocereus polyrhizus (a) purified juice (B) Aqueous ethanolic assay (C) ethanolic assay (D) red beet (a) betanin, (a )isobetanin, (b)phyllocactin, (b')isophyllocactin, (c) hylocerenin, (c ) isohylocerenin HPLC profile of concentrated betacyanin extract obtained through enzymatic treatment with different dosage of enzyme (0.1%-2%); from(a-f) Correlation between enzyme concentration and predominant betacyanin pigments Relative peak area changes (%) of betanin, isobetanin, phyllocactin and total peak areas in extracted colorants Uv/vis spectrum of color extracts made from the Hylocereus polyrhizus Hue angle of different concentrated betacyanin extract (Hylocereus polyrhizus) compare to commercial red beet at chroma level Mean values of lightness(l * ), chroma (C * ), hue(h o ) and betacyanin concentration(mg/l) of concentrated betacyanin colorants obtained from different assays Two-dimensional color space of the betacyanin-based food colorants studied 123 xix

21 LIST OF ABBREVIATION ANOVA : Analysis of Variance AOAC : Association of Official Analytical Chemists CFR : Code of Federal Regulations CIE : International Commission on Illumination, Vienna DOPA : Dihydroxyphenylalanine RGB : Color s Additive Primaries; Red, Green, Blue E-162 : Beetroot Red; Betanin EU : European Union FDA : Food and Drug Administration HPLC : High-Performance Liquid Chromatography mg : milligram min : minute ml : milliliter M ± SD : Mean ± standard deviation nm : Nanometer TS : Tinctorial strength USD : U.S. Dollars UV : Ultra Violet ε : Molar absorbance xx

22 CHAPTER I INTRODUCTION The development of foods with an attractive appearance is an important goal in the food industry. Increasingly, food producers are turning to natural food colors, since certain artificial color additives have demonstrated negative health issues following their consumption (Griffiths, 2005). Because of the deficiencies of existing natural food colorants, the demand for natural pigments is repeatedly raised by the food industry. This demand can be fuelled by research to offer a more natural-healthy way of coloring foods and provide a clean label declaration. Therefore, part of plant pigment research is seeking new sources of pigments. This will not only directed in finding natural alternatives for synthetic dyes, but also discover new procedures for the pigment production. The diversity of tropical and subtropical vegetation offers a promising range of unknown plant compounds that might prove applicable for the needs of humans. The most common plant pigments are carotenoids, chlorophylls, anthocyanins and betalains. Most research has been focused on carotenoids and anthocyanins but betalains have recently gained interest in food science. Commonly broad arrays of anthocyanincontaining extracts are used for food coloring purposes; so far, there is only one single betalainic source that has been extensively used in the food industry worldwide. Compared to anthocyanins, betalains are ideal for coloring low-acid foodstuff as they maintain their color over a wide array of ph, from 3 to 7. The most important betalainic sources for natural red coloring are selected varieties of red beet, commercial 1

23 preparations of which are mainly composed of the red-purple betanin and its C15-isomer isobetanin. Betacyanins are important constituents of betalain pigments. They are the main compounds associated with the red color exhibited by flowers, fruits, and other plant tissues. Red beet has been established in the market as the oldest and most abundant red food colorant, called betanin, that is known as E-162 in the European Union (Castellar et al., 2003) and as in the chapter 21 of the Code of Federal Regulations (CFR) section of the Food and Drug Administration (FDA) in the USA (Griffiths, 2005). Practically it has been used to color foods such as yoghurt, confectionery, ice creams, syrups, sausages and processed meats. However, the typical earthy flavor caused by geosmin and high nitrate concentrations associated with the formation of carcinogenic nitrosamines (Stintzing and Carle, 2004a; Strack et al., 2003) may affect its commercial use. Furthermore, the risk to carry-over earth-bound microorganisms from the raw material in red beet is a crucial point (Herbach et al., 2006a; Stintzing and Carle, 2004). Thereby, there is a high demand for other alternative compounds that can be substitute for red beet. In recent years fruits from the Cactaceae have been introduced as a promising betalain source. The fruits of this family plant displayed a broader color spectrum and were devoid of the mention drawbacks in red beet (Stintzing and Carle, 2007). Selected species of cactus fruits recognized as purple-fleshed Hylocereus polyrhizus have very recently been suggested as promising betacyanin source. The primary findings on the pigment potential and the scarce knowledge about color characteristics 2

24 encouraged more investigations. Preliminary data from different Hylocereus genotype were promising and further investigations are needed for process optimisation with respect to color shade and pigment yield. The main obstacle in these fruits is the pectic substances. Method of degradation of pectic material is necessary to facilitate pigment release, increasing yield and reducing processing wastes. In Malaysia fruits of Hylocereus polyrhizus (genus Hylocereus, Cactaceae) have been cultivated as fruit crop locally. Red color pigments from fruits of H. Polyrhizus could be an interesting possibility as alternative to betanin from red beet. In view of the fact that most natural food colorants are supplied as concentrated juices or extracts and to get a better understanding of how pigment profile and hue are related in making decision for commercial applicability, the present work aimed to: 1. To extract betacyanin pigments from pulp and skin of Hylocereus polyrhizus. 2. To identify the constituents of the betacyanin pigments from pulp of Hylocereus polyrhizus. 3. To evaluate the effect of enzyme treatment on yield of betacyanin constituent. 4. To compare the color of extract from Hylocereus polyrhizus to that of red beet. 3

25 CHAPTER II LITERATURE REVIEW 2.1. Physical Basis of Color Color is a mental response to the stimulus that a visible radiation produces on the retina, which is transmitted to the brain by the optical nerve (Wandell, 1985). It is introduced as a psychophysical concept which is related at the same time to the psychology of the observer and physiology of vision and the spectral radiant energy of a source of light (Purve and Lotto, 2002). Color is associated with light waves and their wavelength distributions. Light, the visible part of the electromagnetic spectrum, consist of waves (Fig. 2.1 A). Each wave is introduced by its wavelength and measured in nanometers. Visible wavelengths are those between the violet and red ends of the spectrum, near 400 and 700 nm, respectively (Fig. 2.1 B). The selection absorption of different amounts of the wavelengths within these limits determines the colors of object (Wandell, 1996). Physically, the color of an object is measured and represented by spectrophotometric curves, which are plots of fractions of incident light (that is reflected or transmitted) as a function of wavelength throughout the visible spectrum relative to a reference (Harold, 2001). Visible spectrum region consist of three dominant colors; red, green and blue that they called additive primaries. Combination of two pure additive primaries produces a subtractive primary (cyan, magenta, and yellow) whereas the combination of all three cause white color (Purves et al., 2002). 4